Antiheparin Proteins Secreted by Human Platelets. Purification, Characterization, and Radioimmunoassay

Abstract

Platelet factor 4 (PF4) and low-affinity PF4 (LA-PF4) were purified to homogeneity from washed human platelets by ZnSO4 precipitation and affinity chromatography on heparin-agarose columns. As shown by double immunodiffusion and specific radioimmunoassays, LA-PF4 and PF4 were immunologically distinct. The levels of PF4 and LA-PF4 antigens in intact platelets were 12.4 and 24.2 µg/109 platelets, respectively. Heparin-neutralizing activity was assayed by measuring the degree of hydrolysis of the chromogenic substrate Bz-***Ile-Glu-Gly-Arg-pNA in the presence of antithrombin III and heparin; 1 mg each of PF4 and LA-PF4 neutralized 17.0 and 2.6 units of beef lung heparin, respectively. Both proteins accounted for 28% of the total heparin neutralizing activity of the platelets. The molecular weight of LA-PF4 as calculated by amino acid analysis and SDS Polyacrylamide gel electrophoresis was 9070 and 7800 daltons, respectively. LA-PF4 shared common antigenic determinants with β-thromboglobulin (βTG) and connective tissue-activating peptide III. The difference between LA-PF4 and βTG was shown by electrophoresis on cellulose acetate at pH 8.6. LA-PF4 migrated in the γ globulin region, in contrast to βTG, which migrated in the β globulin region. The NH2 terminal amino acids of LA-PF4 and βTG were asparagine and glycine, respectively. Partial amino acid sequencing of LA-PF4 showed differences between this protein and βTG. At the NH2-terminal end LA-PF4 had four additional amino acids (Asn-Leu-Ala-Lys); however, residues 5-12 of LA-PF4 and 1-8 of βTG were identical.